Estrogens are a group of hormones that are involved in many critical physiological functions in the human body. Estrogen functions include developing the female sex organs, preparing the breast and uterus for pregnancy and breast feeding after childbirth. Estrogens also play important roles in maintaining proper cardiovascular function and bone density. Estrogens are known to stimulate cell proliferation and may increase a woman's risk of developing cancers, especially breast cancer and uterus cancer.
Estrogens bind to estrogen receptors in target cells to regulate cell functions. Two types of estrogen receptors were discovered in human cells, hER-α and hER-β. They share common protein structures, each possessing three independent but interacting functional domains: the N-terminal domain (A/B domain), the central DNA-binding domain (C domain), and the C-terminal ligand-binding domain (D/E/F domain). The N-terminal domain has a ligand-independent activation function (AF-1), which is involved in interaction with co-activators and transcriptional activation of target genes in the absence of ligands. The DNA binding-domain plays important roles in receptor dimerization and binding to specific DNA sequences. The C-terminal ligand binding-domain mediates ligand binding and has a ligand-dependent transactivation function (AF-2), activating gene transcription in the presence of ligands.
The full-length hER-α was identified as a 66 kDa protein and referred to as hER-α66. hER-α66 contains all three functional domains. A splice variant of hER-α66 was later discovered and named hER-α46. hER-α46 has a molecular weight of about 46 KDa and lacks the N-terminal AF-1 domain of hER-α66. Recently, a novel 36 kDa hER-α variant, hER-α36, was identified. It lacks the N-terminal AF-1 domain and the C-terminal AF-2 domain of hER-α66 (Wang et al., Biochem. Biophys. Res. Commun. 336, 1023-1027 (2005)).
hER-α66 is believed to mediate estrogen-stimulated cell proliferation via transcriptional activation of its target genes. Binding of estrogen to hER-α66 activates the transactivation domain of hER-α66 and thus stimulates the expression of downstream target genes and eventually leads to cell proliferation. hER-α46 was found to mediate membrane-initiated and estrogen-stimulated rapid NO synthesis (Li et al., Proc. Natl. Acad. Sci. USA 100: 4807-4812 (2003)). It was also shown that hER-α46, that lacks the AF-1 domain, inhibits the AF-1 activity of hER-α66 (Flouriot, G., EMBO, 19, 4688-4700 (2000)). Since hER-α36 lacks both the AF-1 and AF-2 transcriptional activation domains, it functions as a dominant-negative inhibitor of hER-α66 and hER-β to inhibit both AF-1 and AF-2 functions of hER-α and hER-β. In addition, hER-α36 is localized primarily on the plasma membrane and mediates membrane-initiated mitogenic estrogen signaling that stimulates cell proliferation. (Wang et al., Biochem. Biophys. Res. Commun. 336, 1023-1027 (2005); Wang et al., Proc. Natl. Acad. Sci. U.S.A. 103: 9063-9068 (2006)).
Extensive studies have shown that estrogen signaling is mediated via the classic nuclear transcriptional activation pathways as well as the non-classic membrane-initiated signaling pathways. It seems that hER-α66 and hER-α46 function primarily in the nucleus while hER-α36 functions mainly outside of the nucleus.
It was also shown that hER-α36 lacks Helix 8-12 of the ligand-binding domain of the original hER-α66, which totally changes the ligand binding specificity of hER-α36. Thus, hER-α36 may bind to different ligands from hER-α66 and hER-β.
Epimedium plants contain estrogen-like compounds that act either as agonists or antagonists. Several different species of Epimedium were used as herbal medicines in Traditional Chinese medicines for thousands of years. Epimedium herbs were used to treat a wide variety of diseases such as impotence, spermatorrhea, urination problems, lassitude and soreness of loin and knees, infertility in women, amenorrhea, geriatric depression, rheumatic arthritis, cardiovascular failure, hypertension, chronic bronchitis, angina pectoris etc. (Wu et al., Chemical and pharmacological investigations of Epimedium species: a survey, Prog Drug Res. 60:1-57 (2003)). It was reported that mixture extracted from Epimedium herbs may also reduce the risk of hormone-related cancers (U.S. 20030170292). However, it is not clear which ingredients of Epimedium herbs may have that function.
Over twenty species of Epimedium have been found and characterized and more than 130 different compounds have been identified from Epimedium plants (Wu et al., Chemical and pharmacological investigations of Epimedium species: a survey, Prog Drug Res. 60:1-57 (2003)). The compounds from Epimedium plants are mainly flavonoid glycodies, flavones and icarisides. Examples of compounds isolated from Epimedium plants are apigenin, brevicornin, icariin, kaempferol, luteolin, quercetin and so on.
Icariin is a flavonoid glycoside compound isolated from Epimedium plants. Icariin was shown to have no effect on cell proliferation in vitro. However, two in vivo metabolites of icariin, icaritin and desmethylicaritin, were found to significantly increase proliferation of breast cancer cells (Liu et al., Pharmazie 60: 120-125 (2005); Wang et al., Europ. J. Pharm. 504: 147-153 (2004); Ye et al., Phytomedicine 12: 735-741 (2005); CN03129242).
As estrogen and estrogen receptor related diseases continue to affect many individuals, there remains an urgent need to discover novel compounds and methods useful to prevent and/or treat such diseases.